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Background The role of the pulmonary microbiome in sarcoidosis is unknown. The objectives of this study were the following: (1) examine whether the pulmonary fungal and bacterial microbiota differed in patients with sarcoidosis compared with controls; (2) examine whether there was an association between the microbiota and levels of the antimicrobial peptides (AMPs) in protected bronchoalveolar lavage (PBAL). Methods Thirty-five sarcoidosis patients and 35 healthy controls underwent bronchoscopy and were sampled with oral wash (OW), protected BAL (PBAL), and left protected sterile brushes (LPSB). The fungal ITS1 region and the V3V4 region of the bacterial 16S rRNA gene were sequenced. Bioinformatic analyses were performed with QIIME 2. The AMPs secretory leucocyte protease inhibitor (SLPI) and human beta defensins 1 and 2 (hBD-1 and hBD-2), were measured in PBAL by enzyme-linked immunosorbent assay (ELISA). Results Aspergillus dominated the PBAL samples in sarcoidosis. Differences in bacterial taxonomy were minor. There was no significant difference in fungal alpha diversity between sarcoidosis and controls, but the bacterial alpha diversity in sarcoidosis was significantly lower in OW (p = 0.047) and PBAL (p = 0.03) compared with controls. The beta diversity for sarcoidosis compared with controls differed for both fungi and bacteria. AMP levels were significantly lower in sarcoidosis compared to controls (SLPI and hBD-1: p < 0.01). No significant correlations were found between alpha diversity and AMPs. Conclusions The pulmonary fungal and bacterial microbiota in sarcoidosis differed from in controls. Lower antimicrobial peptides levels were seen in sarcoidosis, indicating an interaction between the microbiota and the innate immune system. Whether this dysbiosis represents a pathogenic mechanism in sarcoidosis needs to be confirmed in experimental studies.

Background Dystroglycanopathy (α-DG) is a relatively common, clinically and genetically heterogeneous category of congenital forms of muscular dystrophy (CMD) and limb-girdle muscular dystrophy (LGMD) associated with hypoglycosylated α-dystroglycan. To date, mutations in at least 19 genes have been associated with α-DG. One of them, GMPPB, encoding the guanosine-diphosphate-mannose (GDP-mannose) pyrophosphorylase B protein, has recently been associated with a wide clinical spectrum ranging from severe Walker-Warburg syndrome to pseudo-metabolic myopathy and even congenital myasthenic syndromes. We re-sequenced the full set of known disease genes in 73 Italian patients with evidence of either reduced or nearly absent α-dystroglycan to assess genotype-phenotype correlations in this cohort. We used innovative bioinformatic tools to calculate the effects of all described GMPPB mutations on protein function and attempted to correlate them with phenotypic expressions. Results We identified 13 additional cases from 12 families and defined seven novel mutations. Patients displayed variable phenotypes including less typical pictures, ranging from asymptomatic hyperCKemia, to arthrogryposis and congenital clubfoot at birth, and also showed neurodevelopmental comorbidities, such as seizures and ataxic gait, as well as autism-spectrum disorder, which is seldom described in clinical reports of dystroglycanopathies. We also demonstrated that few mutations recur in the Italian GMPPB-mutated population and that alterations of protein stability are the main effects of GMPPB missense variants. Conclusion This work adds to the data on genotype-phenotype correlations in α-DG and offers new bionformatic tools to provide the conceptual framework needed to understand the complexity of these disorders. Electronic supplementary material The online version of this article (10.1186/s13023-018-0863-x) contains supplementary material, which is available to authorized users.

In the post-genomic era, one of the major challenges is the identification of alleles directly responsible for phenotype variation among different genotypes within the same species. Tomato is a model crop for understanding the development and ripening of climacteric fleshy fruits, and it is also known to be an important source of health-promoting compounds. In addition, cultivated tomato germplasm shows a high phenotypic variation despite its very low genetic diversity. Toward the identification of sequence variations responsible for stress tolerance, high fruit quality and long shelf life, we re-sequenced the genomes of two traditional landraces grown in the Campania region (Southern Italy). Crovarese, belonging to the Corbarino type (COR), and Lucariello (LUC) are typically grown under low water regimes and produce highly appreciated fruits, which can be stored up to 4-8 months. We generated 65.8M and 56.4M of paired-end 30-150 bp reads with an average insert size of 380 bp (± 52bp) and 364 bp (± 49bp) for COR and LUC, respectively. A referenceguided assembly was performed using 'Heinz 1706' as a reference genome. We estimated a mean coverage depth of ~15X for COR and 13X for LUC. Comparing the genomes of COR and LUC with that of 'Heinz 1706' we found a similar distribution of SNPs (68.8% vs. 69.9%, respectively), small deletions (8.9% vs. 8.6%) and small insertions (22.1% vs. 21.3%). Through a de novo assembly of the unmapped reads we identified 29 and 36 new contigs in COR and LUC, respectively. The new contigs could be assigned to the chromosomes thanks to the use of a splitread approach. On average, the contigs inserted in COR were 654bp, whereas those inserted in LUC were 616bp. Using custom RNA-seq data, a total of 43054 and 44576 gene loci were annotated in COR and LUC, corresponding to 62369 and 65094 transcripts, respectively. Among the genes showing a similar structure in COR and LUC compared to 'Heinz 1706', we identified ~2000 and 1700 SNPs causing potentially disruptive effects on the function of 1371 and 1201 genes in COR and LUC, respectively. Interesting GO categories highly represented in genes affected by sequence changes were identified. Major variations were present in stress-responsive genes as well as in fruit quality and development-related genes. From a practical perspective, the identified SNPs and InDels are candidate polymorphisms to track DNA variations associated to key traits of economic interest.

Tomato is a high value crop and the primary model for fleshy fruit development and ripening. Breeding priorities include increased fruit quality, shelf life and tolerance to stresses. To contribute towards this goal, we re-sequenced the genomes of Corbarino (COR) and Lucariello (LUC) landraces, which both possess the traits of plant adaptation to water deficit, prolonged fruit shelf-life and good fruit quality. Through the newly developed pipeline Reconstructor, we generated the genome sequences of COR and LUC using datasets of 65.8M and 56.4M of 30-150bp paired-end reads, respectively. New contigs including reads that could not be mapped to the tomato reference genome were assembled, and a total of 43, 054 and 44, 579 gene loci were annotated in COR and LUC. Both genomes showed novel regions with similarity to Solanum pimpinellifolium and Solanum pennellii. In addition to small deletions and insertions, 2, 000 and 1, 700 single nucleotide polymorphisms (SNPs) could exert potentially disruptive effects on 1, 371 and 1, 201 genes in COR and LUC, respectively. A detailed survey of the SNPs occurring in fruit quality, shelf life and stress tolerance related-genes identified several candidates of potential relevance. Variations in ethylene response components may concur in determining peculiar phenotypes of COR and LUC.

Nitrogen (N) is a critical macronutrient for plants and its utilization is fundamental to crop productivity. In the last decades, N fertilizers have been extensively used to increase both grain yield and grain protein content in wheat, helping support the human population growth while causing a serious pollution problem. Wheat is one of the most used cereal crops for the important amount of proteins it provides in human diet, and durum wheat (Triticum turgidum ssp. durum) is particularly important in the Mediterranean region for the production of pasta and other typical dishes. In order to use gene knowledge to improve N-use efficiency, it is crucial to increase the understanding of how this crop responds to growth under different N conditions. Here, high-throughput sequencing was used to provide a determination of transcript levels of N-starvation-induced genes. We constructed 24 cDNA libraries from roots, leaves/stems, flag leaf and spike of durum wheat cultivar Svevo plants grown under N-deficient or standard conditions during the grain filling stage. A RNA-Seq experiment on three biological replicates for each tissue provided a large amount of sequencing data, which were used for a differential expression analysis. A total of 4968 transcripts were found differentially expressed between treated and control samples. Among them, 3270 belonged to root tissues, while the other 1698 were genes found in leaves/stems, flag leaf and spike tissues. Results highlighted that 3057 genes were differentially expressed uniquely in root tissues, 681 in leaves/stems, 298 in flag leaf and 234 only in spike tissues. The Gene Ontology analysis revealed a number of categories involved in N-stress in all organ types, the most frequent down-regulated transcripts functioned for thiamin biosynthetic and metabolic processes, photosynthesis, protein processing, localization and maturation, photorespiration, respiratory chain. Other categories such as CTP biosynthetic and metabolic processes, DNA synthesis, and amino acid metabolism were also represented. Selected genes were analysed in quantitative PCR in order to confirm their expression levels. Our results provide valuable information about N-starvation-responsive genes involved in different genetic pathways and concerning several plant organs of durum wheat. This data will help shed light on signal transduction pathways related to N-utilization and will be useful for identifying candidate genes for crop breeding.

Considered as one of the most important horticultural crops worldwide, tomato (Solanum lycopersicum L.) has had a huge agronomical success in the Mediterranean area, where numerous local landraces which show great phenotypic diversity were selected. Among the several landraces grown in Southern Italy and analyzed by our research group, two of these, Lucariello (LUC) and Crovarese (CRO) showed interesting phenotypes in field trials repeated over several years. LUC and CRO displayed a high tolerance to drought stress, and produced fruits of high quality and long shelf life despite the low water input regimes, making them very attractive economically and scientifically. To identify the components involved in drought stress responses, we pursued a transcriptome study on CRO and LUC plants subjected to drought stress by water withholding for 13 days. Our trascriptome sequencing was done in conjunction with a whole-genome resequencing project of CRO and LUC, which allowed us first to use the specific genomes of CRO and LUC as reference for the bioinformatic pipeline, and to merge the genomic data with drought stress-induced gene expression variations. Using RNA sequencing on leaf samples, we identified 2954 and 2015 genes up-regulated upon stress in CRO and LUC, respectively. By contrast, 3912 genes were down- regulated in CRO and 2671 in LUC. Gene Onthology (GO) enrichment analysis on up-regulated and down-regulated genes shared between CRO and LUC revealed that several of the enriched categories were stress-related. Comparison of the RNA-seq results with polymorphic genes identified within the resequencing project revealed 300 drought-responsive genes common to CRO and LUC which carried sequence polymorphisms with a potentially disruptive impact on their function. Among these, 101 were up- and 199 down-regulated genes in drought stress conditions. The polymorphisms identified included nonsense mutations generating premature stop codons in a number of expressed genes. The pectinase family involved in cell wall organization showed a striking number of members, 42 whose expression was repressed by water deprivation. Among these, 3 genes were selected (Solyc01g079180, Solyc05g014000, Solyc09g075460) for expression analysis in CRO, LUC and in a commercial genotype (Red Setter, RS) during two cycles of stress and a recovery phase. Results highlighted an opposite behavior of these genes between the traditional landraces and RS during the second cycle of stress, where an expression inhibition was observed in CRO and LUC compared to an upregulation in RS. The observed downregulation of the pectinase genes could be part of a genotype-specific response to drought, leading to reduced degradation of pectin structure in the cell wall to respond to water scarcity. Altogether, the gene expression data presented here contribute to an understanding of the drought tolerance displayed in ? the field by CRO and LUC and to the identification of genes putatively involved in the stress adaptation features of these traditional landraces.

In common bean (Phaseolus vulgaris L.), lectin (phytohemagglutinin, PHA) and the lectin-related storage proteins arcelin (ARC) and ?-amylase inhibitor (?-AI) have evolved in a single complex genetic locus, called APA locus. Although these proteins are considered true storage proteins, several experimental data suggest a role in protection of the seed from two bruchid beetles, the bean weevil (Acanthoscelides obtectus Say) and the Mexican bean weevil (Zabrotes subfasciatus Bohemian). So far, seven ARC variants have been identified, all in wild accessions, however, only those containing ARC-4 or ARC-8 proteins were reported to be resistant to both species [1]. Molecular evolutionary analysis on this multigene family indicated that gene duplication with subsequent diversification has played a very important role in the evolution of this locus [2, 3]. We present biochemical and immunological data showing the variability of APA components in a collection of wild accessions, many of which identified as ARC variants. To shed light on the molecular structure and evolution of the APA locus, 23 BAC clones covering the whole APA locus of the wild accession G12946, which contains the most complex APA locus (Arc/PHA/?-AI) were submitted for paired-end read sequencing on an Illumina MiSeq platform (San Diego, CA, USA). This locus is over 500 Kbp, i.e. ten times larger than that found in published bean genomes (Andean G19833 and Mesoamerican Bat93). Sequencing data show that, beside genes coding for known APA proteins, many repetitive DNA sequences, microsatellites and retrotrasposons, together with several interrupted lectin and arcelin related sequences were also found at this locus, suggesting that it arised through a "birth and death" model of evolution, which is particularly true for the arcelin type sequences. Although many efforts have been made, a successful breeding of the bruchid resistance trait into cultivated genotypes has hardly been achieved. The availability of the sequence of this APA locus will provide a useful tool to develop new markers to help in breeding for such purpose.

Drought is one of the major abiotic stresses affecting plant growth and productivity. Among horticultural crops, tomato (Solanum lycopersicum) is generally classified as susceptible to drought stress. However, several local landraces adapted to arid Mediterranean conditions are successfully cultivated in low water input regimes. In order to identify the components involved in drought stress responses, we pursued an integrated approach where several biochemical and physiological parameters were evaluated together with transcriptome sequencing in two landraces ( Lucariello and Crovarese) from the Campania Region. Plants were grown in greenhouse and subjected to drought stress by water withhold for 13 days. Stomatal conductance and photosynthetic efficiency parameters showed similar profiles in both genotypes, with values greatly reduced after prolonged water withhold, indicative of a severe water stress condition. Similarly, ABA and proline levels greatly increased due to the stress treatment. Using RNA sequencing on leaf samples, we identified 2851 and 1867 genes upregulated upon stress in Crovarese and Lucariello, respectively. Furthermore, 4000 genes were downregulated in Crovarese and 2642 in Lucariello. Extensive overlapping was observed, with 1419 up- and 2297 down-regulated genes common to both genotypes. Gene Onthology (GO) enrichment analysis carried out on up-regulated and down-regulated genes revealed that several of the enriched categories were stress-related. A k-means clusterization was carried out. We identified in both genotypes 8 clusters (4 in upregulated dataset and 4 in downregulated dataset) of genes with similar expression profile. A new GO enrichment aimed at understanding the fuction of the clusters identified classes, which were peculiar for specific clusters. Among the genes showing the highest up-regulation in the stress treatment several members of the Late Embryogenesis Abundant protein (LEA) family were identified. This family was therefore selected for further studies. LEA proteins are a group of proteins that accumulate in response to cellular dehydration in many organism. Using multiple approaches for the discovery of LEA genes, we could identify 37 LEA proteins encoded in the tomato genome. We then built a dendrogram, which allowed the assignment of each gene to one of the known 5 LEA sub-family. Comparison of the dendrogram structure with transcriptome results highlighted that the 2 subfamilies "LEA 4" and "Dehydrin" in particular were upregulated following drought treatments in leaves. Altogether, our results contribute a broad understanding of the physiological, biochemical and molecular mechanisms employed by tomato to respond to water deprivation.